Recombinase polymerase amplification assays for the identification of pork and horsemeat

Kissenkötter, Jonas; Böhlken-Fascher, Susanne; Forrest, Matthew S.; Piepenburg, Olaf; Czerny, Claus-Peter; Wahed, Ahmed Abd El

doi:10.1016/j.foodchem.2020.126759

Cited by 36 publications

(25 citation statements)

References 26 publications

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“…The standard genomic DNA of goat and donkey were provided by Eurofins GeneScan Technologies GmbH (Freiburg, Germany). According to the German Federal Office of Consumer Protection and Food Safety, 0.1% considered as the lower limit of detection of meat contamination (German Food and Feed Code §64 (LFGB)) [ 12 ], therefore, using a sensitive balance, 50 mg of pork meat was spiked with 0.5 mg of cattle, sheep, horse, chicken, turkey, duck, and rabbit meat. Since, meat source from goat and donkey were not available, genomic DNA of both species equivalence to 10 3 molecules were added to the mix.…”

Section: Methodsmentioning

confidence: 99%

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Flesh ID: Nanopore Sequencing Combined with Offline BLAST Search for the Identification of Meat Source

Kissenkötter

Böhlken-Fascher

Wahed

2020

Foods

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Detection of animal species in meat product is crucial to prevent adulterated and unnecessary contamination during processing, in addition to avoid allergy and religious consequences. Gold standard is the real-time PCR assays, which has a limited target capability. In this study, we have established a rapid sequencing protocol to identify animal species within hours. Sequencing was achieved by nanopore sequencing and data analysis via offline BLAST search. The whole procedure was conducted in a mobile suitcase lab. As per national and international regulations, the developed assay detected adulteration of pork meat with 0.1% of horse, chicken, turkey, cattle, sheep, duck, rabbit, goat, and donkey. The developed test could be used on-site as a rapid and mobile detection system to determine contamination of meat products.

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Section: Methodsmentioning

confidence: 99%

“…The gold-standard in the authentication process of meat and meat products is the real-time PCR [ 10 ]. Recently, many isothermal amplification assays were established [ 11 , 12 ]. However, these molecular tests are limited to few targets and are unable to identify species outside the narrow target range.…”

Section: Introductionmentioning

confidence: 99%

Flesh ID: Nanopore Sequencing Combined with Offline BLAST Search for the Identification of Meat Source

Kissenkötter

Böhlken-Fascher

Wahed

2020

Foods

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“…Recombinase polymerase amplification (RPA) is generally used to detect meat adulterants (chicken, duck, pig, etc.) with fluorescent and color detection [44,45] or a lateral flow test [46][47][48][49][50]. Other isothermal amplification methods have been employed for designing adulteration tests, including rolling circle amplification for horse-meat identification [51] and single primer-triggered isothermal amplification for chicken-meat detection [52].…”

Section: Introductionmentioning

confidence: 99%

Rapid Full-Cycle Technique to Control Adulteration of Meat Products: Integration of Accelerated Sample Preparation, Recombinase Polymerase Amplification, and Test-Strip Detection

et al. 2021

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Verifying the authenticity of food products is essential due to the recent increase in counterfeit meat-containing food products. The existing methods of detection have a number of disadvantages. Therefore, simple, cheap, and sensitive methods for detecting various types of meat are required. In this study, we propose a rapid full-cycle technique to control the chicken or pig adulteration of meat products, including 3 min of crude DNA extraction, 20 min of recombinase polymerase amplification (RPA) at 39 °C, and 10 min of lateral flow assay (LFA) detection. The cytochrome B gene was used in the developed RPA-based test for chicken and pig identification. The selected primers provided specific RPA without DNA nuclease and an additional oligonucleotide probe. As a result, RPA–LFA, based on designed fluorescein- and biotin-labeled primers, detected up to 0.2 pg total DNA per μL, which provided up to 0.001% w/w identification of the target meat component in the composite meat. The RPA–LFA of the chicken and pig meat identification was successfully applied to processed meat products and to meat after heating. The results were confirmed by real-time PCR. Ultimately, the developed analysis is specific and enables the detection of pork and chicken impurities with high accuracy in raw and processed meat mixtures. The proposed rapid full-cycle technique could be adopted for the authentication of other meat products.

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“…2018; Kissenkotter et al . 2020), has drawn great attention due to its high specificity and sensitivity, constant temperature (37–42°C), rapid detection (20–30 min) and popularization (Wang et al . 2017; Hu et al .…”

Section: Introductionmentioning

confidence: 99%

“…Recently, recombinase polymerase amplification (RPA), as a novel isothermal amplification technology of nucleic acid for the diagnosis of infectious disease in DNA or RNA levels (Law et al 2018;Kissenkotter et al 2020), has drawn great attention due to its high specificity and sensitivity, constant temperature (37-42°C), rapid detection (20-30 min) and popularization (Wang et al 2017;Hu et al 2020). RPA couples isothermal recombinase-driven primer targeting the template with strand-displacement DNA synthesis, which can achieve exponential amplification (Xia et al 2014;Lai and Lau 2020).…”

Section: Introductionmentioning

confidence: 99%

Rapid detection of Mycobacterium tuberculosis based on antigen 85B via real-time recombinase polymerase amplification

Zhang

et al. 2021

Letters in Applied Microbiology

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Tuberculosis (TB), as a common infectious disease, still remains a severe challenge to public health. Due to the unsatisfied clinical needs of currently available diagnostic vehicles, it is desired to establish a new approach for universally detecting Mycobacterium tuberculosis. Herein, we designed a real‐time recombinase polymerase amplification (RPA) technology for identifying M. tuberculosis within 20 min at 39°C via custom‐designed oligonucleotide primers and probe, which could specifically target antigen 85B (Ag85B). Particularly, the primers F4‐R4 produced the fastest fluorescence signal with the probe among four pairs of designed primers in the RPA assays. The optimal primers/probe combination could effectively identify M. tuberculosis with the detection limit of 4·0 copies per μl, as it could not show a positive signal for the genomic DNA from other mycobacteria or pathogens. The Ag85B‐based RPA could determine the genomic DNA extracted from M. tuberculosis with high reliability (100%, 22/22). More importantly, when testing clinical sputum samples, the real‐time RPA displayed an admirable sensitivity (90%, 95% CI: 80·0‐96·0%) and specificity (98%, 95% CI: 89·0‐100·0%) compared to traditional smear microscopy, which was similar to the assay of Xpert MTB/RIF. This real‐time RPA based Ag85B provides a promising strategy for the rapid and universal diagnosis of TB.

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Recombinase polymerase amplification assays for the identification of pork and horsemeat

Cited by 36 publications

References 26 publications

Flesh ID: Nanopore Sequencing Combined with Offline BLAST Search for the Identification of Meat Source

Flesh ID: Nanopore Sequencing Combined with Offline BLAST Search for the Identification of Meat Source

Rapid Full-Cycle Technique to Control Adulteration of Meat Products: Integration of Accelerated Sample Preparation, Recombinase Polymerase Amplification, and Test-Strip Detection

Rapid detection of Mycobacterium tuberculosis based on antigen 85B via real-time recombinase polymerase amplification

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